Lam, TuKiet T.

Alcohol-associated liver disease (ALD) is a growing global health concern, with alcohol-associated hepatitis (AH) leading to the highest morbidity and mortality. Available therapies are limited and often inadequate. Platelets contribute in a variety of ways to liver disease pathogenesis, but their role in AH remains largely unexplored. In this study, we addressed the hypothesis that platelets contribute to pathological inflammation in AH. Using patient samples and a multiomics approach, we found that platelets undergo proinflammatory transcriptomic and proteomic changes in AH, with 2 alarmins, S100A8 and S100A9, being among the top upregulated genes/proteins. Additionally, the abundance of platelet-derived microparticles containing S100A8 and S100A9 in AH patient plasma was increased and correlated with disease severity (assessed by model for end-stage liver disease sodium [MELD-Na]) and endotheliopathy (assessed by ICAM1, CXCL8, and vWF). We mechanistically linked S100A9 with endotheliopathy via crosstalk between primary human liver sinusoidal endothelial cells and primary human monocytes. We also demonstrated that IL-6 upregulates S100A9 in megakaryocytic cells in a JAK/STAT-dependent manner, modeling changes occurring in the bone marrow in patients with AH. Our studies establish proinflammatory platelets as important contributors to AH pathology. Moreover, antiplatelet agents — or, more specifically, S100A9 targeted drugs — are potential therapeutic strategies in AH.

Abstract Glioblastoma (GBM), the most aggressive primary brain tumor in adults, presents significant challenges due to its universal recurrence and limited survival rates, exacerbated when proximal to the lateral ventricles (LV). The subpopulation of brain tumor-initiating cells (BTICs) plays a pivotal role in tumor initiation and invasiveness, interacting with the microenvironment, particularly the cellular components in the subventricular zone (SVZ), like Neural Progenitor Cells (NPCs). Mechanisms of intercellular communication present in the SVZ include paracrine, autocrine, direct cell contact, and more recently extracellular vesicles (EVs). EVs are membrane-bound particles released by cells that are central to intercellular communication. They carry bioactive proteins, RNA, and other molecules that influence recipient cell behavior. This study investigates the role of EVs in GBM’s communication with non-cancer cells. Our findings indicate that BTIC-derived EVs promote migration and proliferation (*p < 0.02, ****p <0.001) in NPCs in vitro, highlighting the EVs’ role in altering the microenvironment. Additionally, we leverage a multi-omic approach to profile the small RNA and protein cargo of BTIC-EVs while also evaluating changes in the transcriptome of NPCs after BTIC-EV treatment. To identify the protein cargo contained in GBM-derived EVs, we utilized the innovative proximity-labeling system TurboID. We successfully induced TurboID expression in primary-cultured human BTICs from GBM patients, enabling the unbiased biotinylation of proteins in EVs. This method marks the first implementation of TurboID for unbiased global labeling of EV protein cargo in primary GBM cells. We identified unique miRNAs to be regulators of NPC’s transcriptome, and ontogenic analysis revealed upregulation of pathways involved in maintaining stem-like state, and upregulating PI3k-AKT, mTOR, and WNT signaling. This approach facilitates the investigation of EV-mediated communication and potential therapeutic targets, contributing to the understanding of GBM’s complex interactions with the brain’s microenvironment and the identification of biomarkers for improved diagnosis and treatment response.

ABSTRACT Glioblastoma (GBM), the most aggressive primary brain tumour in adults, presents significant challenges due to its universal recurrence and limited survival rates. A key driver of GBM progression is the subpopulation of brain tumour‐initiating cells (BTICs), which contribute to therapy resistance and interact with the tumour microenvironment, particularly the cellular components in the subventricular zone (SVZ). Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying bioactive molecules, such as proteins and RNAs, that can modulate the behaviour of recipient cells. This study investigates the role of EVs in GBM's communication with non‐cancer cells. We utilised the proximity‐labelling system TurboID to achieve global and unbiased labelling of proteins within GBM‐derived EVs. By inducing TurboID expression in primary‐cultured human BTICs from GBM patients, we achieved efficient biotinylation of EV proteins without compromising vesicle integrity, and performed proteomic analysis of biotinylated proteins, which revealed a diverse cargo within BTIC‐EVs. Our method marks the first implementation of TurboID for unbiased global labelling of EV protein cargo in primary cells. This approach facilitates the investigation of EV‐mediated communication and potential therapeutic targets, contributing to the understanding of GBM's complex interactions with the brain's microenvironment and identification of biomarkers for improved diagnosis and treatment response.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor–initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.